Process for obtaining aroma components and aromas from their precursors of a glycosidic nature, and aroma components and aromas thereby obtained

ABSTRACT

A method is disclosed for obtaining aroma components and aromas from their glycosidic precursors containing a β-apioside with the use of β-apiosidase.

This application is a continuation of application Ser. No. 08/276,779, filed Jul. 18, 1994 (U.S.Pat. No. 5,573,926, Issue Date: Nov. 12, 1996), which is a continuation of application Ser. No. 07/681,520 filed Jul. 5, 1991 filed as PCT/EP 90/01545, Sep. 7, 1990, now abandoned.

The present invention relates to a process for obtaining aroma components or aromas from their precursors of a glycosidic nature, as well as to the aroma components and aromas obtained by this process.

TECHNICAL BACKGROUND

In some wine varieties, such as muscats, the aroma compounds exist in two forms, free and bound. The free fraction consists of odoriferous volatile substances, chiefly terpenols. The bound fraction contains precursors of terpenols, especially non-odoriferous diglycosides, formed from α-L-rhamnopyranosyl-β-D-glucopyranosides (designated Rha-Glc), from α-L-arabinofuranosyl-β-D-glucopyranosides (designated Ara-Glc) and from β-D-apiofuranosyl-β-D-glucopyranosides (designated Api-Glc), in which the glucopyranose provides the link between the terpene residue (designated Terp) and the disaccharide, according to the formulae:

    Rha(1→>6)Glc-Terp

    Ara(1→>6)Glc-Terp

    Api(1→>6)Glc-Terp

The aroma fraction in the form of precursors is most often much larger than the free aroma fraction (typically by a factor of 3 to 10), and it can reach high concentrations, of the order of a few milligrams per litre.

Taking into account, in addition, the particularly low threshold of olfactory perception and the aromatic quality of terpene alcohols, there is, in these vine varieties, a most important unexploited aroma potential.

The terpene glycosides, present in the juice, may be hydrolysed using commercial enzyme preparations with a wide variety of specifications. The enzymatic liberation of terpenols, which reflects the free natural aroma of the fruit more faithfully than that revealed by thermal hydrolysis at the pH of the juice, is hence possible; however, the control of this liberation for an industrial exploitation of the aroma potential presupposes that the glycosidases responsible for the hydrolyses are defined and their mechanism of action established.

BACKGROUND ART

Strauss et al. (in Parliament et al., Bioceneration of aromas, 1986, American Chemical Society, Washington, D.C., pp. 222-239) discuss the importance of monoterpenes in grape and wine flavor and the extent to which these compounds contribute to varietal character which is especially significant in winemaking.

Pisarnitskii, A. F. (Chemical Abstract 128650c, 1971, Columbus, Ohio) discloses a method of imparting odors to wines via the enrichment of the product with substances in the pulp to increase the activity of pulp enzymes that are responsible for enzymatic synthesis of the aromatic substances present in the product and to impart the desired arome to the finished product.

Drawert, F. et al. (J. Agric. Food Chem., 1978, vol. 26, no. 3, pp. 765-766) disclose the finding of the monoterpenes citronellol, linalool and geraniol in the oderous constituents produced by the yeast Kluyveromyces lactis in aerobic submersed culture. By changing of the culture conditions, it was possible to influence the biosynthesis of citronellol in K. lactis.

SUMMARY OF THE INVENTION

The present invention is based on a demonstration of the mechanism of enzymatic hydrolysis, such mechanism being of the sequential type.

Thus, the process according to the present invention for obtaining aroma components and aromas from their precursors of a glycosidic nature is characterized in that:

in a first stage, an enzymatic hydrolysis of a glycosidic substrate containing at least one of the said precursors is performed with at least one enzyme chosen in accordance with the structure of the said precursor, to liberate the corresponding monoglucosides by cleavage at a glycoside bond;

in a second stage, an enzymatic hydrolysis of the product of the first stage is performed with at least one enzyme other than or identical to that/those of the first stage and designed to liberate the aroma components and aromas by cleavage of the aglycone-carbohydrate link bond.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Chromatographic profile of α-rhamnosidase and β-glucosidase activities.

FIG. 2: Chromatographic profile of α-arabinosidase, α-rhamnosidase and β-glucosidase activities.

FIG. 3: Chromatographic profile of α-arabinosidase, α-rhamnosidase and β-glucosidase activities following DEAE-Sepharose CL-6B ion exchange chromatography.

FIG. 4: Chromatographic profile of α-arabinosidase activity following concanavalin A-Ultrogel affinity chromatography.

FIG. 5: Chromatographic profile of α-arabinosidase activity as a function of pH and temperature.

FIG. 6: TLC monitoring of the enzymatic hydrolysis of synthetic Ara-Glc-pNP and Rha-Glc-pNP substrates.

FIG. 7: TLC monitoring of the enzymatic hydrolysis of a glycosidic extract.

FIG. 8: Monitoring by GC of the enzymatic hydrolysis of a glycosidic extract by α-arabinosidase±β-glucosidase.

FIG. 9: Monitoring by GC of the sequential hydrolysis of a glycosidic extract by α-rhamnosidase+β-glucosidase.

FIG. 10: HPLC chromatogram of terpene glycosides.

FIG. 11: GC Chromatograms of glycosidic extracts from the must of Muscat de Frontignan grapes (A); enzymatically treated with Hemicellulase (B); enzymatically treated with Klerzyme 200 (C).

FIG. 12: GC Chromatograms of glycosylated aroma fractions from the musts of naturally sweet wines: (A) enzymatically treated; (B) non-enzymatically treated; and dry wines: (C) enzymatically treated; (D) non-enzymatically treated.

DETAILED DESCRIPTION OF THE INVENTION

"Substrate" is understood to mean any substance containing a precursor of a glycosidic nature of an aroma component or aroma.

By way of an example, the glycosidic precursor may be a vegetable material derived from grapes, such as grape juices, wines and their derivatives, for example all drinks containing wine, grape juice or a related substance, as well as by-products of the vinification of aromatic vine varieties, especially muscats. The four components of the enzyme system needed for the hydrolysis of the terpene glycosides of grapes to liberate terpenols, odoriferous volatile substances, are now known according to the invention: an α-arabinosidase, an α-rhamnosidase and a β-apiosidase for the first stage, and β-glucosidase for the second stage. The work leading to the present invention has enabled, in effect, to be concluded that the hydrolysis of terpene diglycosides does not proceed from the action of a single glycosidase but, on the contrary of several, operating in pairs according to the following two-stage sequential mechanism:

Stage 1:

Action of an α-arabinosidase, an α-rhamnosidase and a β-apiosidase liberating the corresponding terpene monoglucosides by cleavage at the (1→>6) glycoside bond.

Stage 2:

Action of a β-glucosidase providing for the liberation of the terpenols by cleavage of the terpene aglycone-carbohydrate link bond.

    ______________________________________                                         Stage 1         Stage 2                                                        ______________________________________                                         Ara (1 → 6) Glc-Terp                                                      α-arabinosidase                                                          Rha (1 → 6) Glc-Terp → Glc-Terp → Terpenol                                            α-rhamnosidase β-glucosidase                                        Api (1 → 6) Glc-Terp                        β-apiosidase                                                            ______________________________________                                    

The last key enzyme, namely the β-glucosidase, on which the final liberation of the terpenols depends, preferably exhibits minimal inhibition by glucose, activity at acid pH values and high affinity with respect to the aglycones engaged in the glycosidic substrates, in order for the proposed process to possess complete efficacy in its applications.

According to the invention, the substrates (Ara-Glc-pNP, Rha-Glc-pNP, Api-Glc-pNP, Ara-pNP, Rha-pNP, Api-pNP, Glc-pNP) enabling the activities to be measured, have also been defined, these conditions being essential for the production of the corresponding enzymes.

The process according to the present invention is exemplified by the exploitation of the aroma potential of grapes, the primary plant material being chosen from grape juices, wines and derivatives, as well as by-products of the vinification of aromatic vine varieties, especially but not exclusively muscats.

However, the present invention is not limited to this application. In effect, the mechanism demonstrated is a general mechanism leading to aroma components and aromas which are not necessarily terpenic, and which is applicable to all plant products other than grapes: fruits, fruit derivatives (for example drinks) and fruit by-products; aromatic plants and flowering plants, as well as derivatives and by-products of these plants; other plants such as tea and tobacco; and even plant material originating from in vitro cell cultures; with the following provisos:

(a) that these plant products contain, in sufficient amounts, aroma or perfume precursors of a glycosidic nature, including terpenols and glycosides other than those encoutered in grapes,

(b) that the specific glycosidases correspond to the structure of the glycosidic precursors; and

(c) that the natural medium does not contain inhibitors of the enzymes applied.

As fruits containing glycosides, other than grapes, which can be used in the process of the present invention, apricots, mangos, papayas and passion fruit may be mentioned, inter alia. As a flowering plant, the rose may be mentioned inter alia.

It is also possible to use a glycosidic extract as a glycosidic substrate, or the hydrolysis can alternatively be performed on a natural medium containing the precursors.

Thus, a large number of possibilities are offered for carrying out the process according to the present invention:

working on a natural medium, as described above, the bound aroma is liberated, thereby increasing the aroma of the product itself;

it is also possible to treat a given glycosidic extract (for example of papaya) and to introduce the aroma obtained into another product, for example a drink (grape juice);

it is also possible to introduce one (or more) glycosidic extract(s) (for example papaya extract, marc extract) into a liquid substrate (for example grape juice, natural drinks) and to apply the process according to the invention, thereby liberating the aroma in situ. It is also possible to contrive that the aroma is liberated later, at the desired moment, in a food, a drink or a perfume.

The term "enzyme" defines here any means capable of obtaining the corresponding enzymatic activity. The enzymes employed in the process according to the present invention can be of any origin: bacterial, fungal, yeast, plant, animal or synthetic. Enzymes produced by genetic manipulations using host microorganisms are also encompassed according to the invention.

Microorganisms can thus be modified by techniques known to those versed in the art for producing an enzyme or several enzymes, which can be usable in the process.

The process of the invention makes it possible to obtain, in particular as aroma components or aromas, terpenols such as geraniol, linalool, hydroxylinalools, oxydes of linalool, nerol, citronellol, α-terpineol, nor-isoprenoids compounds (such as hydroxy-3-damascone, 3-oxo-α-ionol), terpene polyols and alcohols such as phenyl ethyl and benzyl alcohol, or the like.

According to the invention, it is also possible, depending on the profile of the aroma bound to the precursors, which can be different from the free aroma, or depending on the specificity of the enzyme used, to envisage the production of a novel aroma.

Knowledge of the mechanism results in the production of enzymes having novel characteristic properties and technological capabilities.

According to a particular embodiment of the process of the present invention, corresponding to the case where the substrate contains only monoglucosides, the enzymatic hydrolysis is performed directly without passing through the first stage.

In the definition of the invention, as mentioned above, it was stated that the process involved a two-stage hydrolysis. However, the invention is in no way limited to the use of two stages with successive additions of separate enzymes. As a variant, it is in effect, perfectly possible, especially if it is the same enzyme or enzymes which liberate(s) the monoglycosides and also the aroma components and aromas, to bring the glycosidic substrate into contact in a single stage with the chosen enzyme or enzymes, the enzymatic hydrolysis then proceeding in at least one reaction phase leading to the desired aromas and/or aroma components. This embodiment of the process of the invention can prove suitable, for example, if at least one microorganism encoded for producing the enzyme or enzymes useful in the reaction is used for the reaction with the glycosidic substrate. In the description which follows, when a first stage and a second stage are referred to in the interest of the convenience of the description, this simply means that the hydrolysis reaction proceeds in several phases, but this in no way means that these reaction phases necessitate separate additions of enzymes.

The following examples are provided as a means of illustrating the instant invention, and are not intended to limit the invention.

EXPERIMENTAL SECTION A. SUBSTRATES

Among the substrates used, p-nitrophenyl β-D-glucopyranoside (Sigma, USA), p-nitrophenyl α-L-arabinofuranoside (Sigma, USA) and p-nitrophenyl α-L-rhamnopyranoside (Extrasynthese, France) are commercially available. Geranyl β-D-apiofuranosyl-β-D-glucopyranoside is an extract of the medicinal plant species Hypoxis acuminata. The other glycosides were synthesized: their synthesis employs three stages, starting with the corresponding peracetylated saccharide.

The first staoe consists in the activation of the anomeric carbon of the terminal carbohydrate group of the peracetylated corresponding saccharide, by introduction, onto this carbon, of a halogen such as chlorine or bromine, or an imidate such as trichloroacetimidate.

Thus, hexaacetyl-α-chlororutinoside is obtained by the action of zinc dichloride and dichloromethyl methyl ether on peracetylated rutin in an inert solvent such as chloroform or methylene chloride, under anhydrous conditions.

Tetraacetyl-α-bromo-D-glucopyranoside and hexaacetyl-α-bromorutinoside are prepared by the action of gaseous hydrobromic acid on pentaaceto-D-glucopyranose and on heptaacetorutinose, respectively, in an inert solvent such as chloroform or methylene chloride, under anhydrous conditions.

The mixture of O-(hexaacetyl α- and -β-rutinosyl) trichloroacetimidates is obtained by the action of hexaacetyl-1-rutinose (obtained by the action of benzylamine or ammonia on heptaacetorutinose in an aprotic solvent such as acetonitrile, tetrahydrofuran or ethyl ether) on trichloroacetonitrile, in the presence of a base, for example potassium carbonate or sodium hydride, in an aprotic solvent such as methylene chloride, chloroform or diethyl ether and under anhydrous conditions. O-[Hexaacetyl-6-O-(α-L-arabinofuranosyl)-α- and -β-D-glucopyranosyl] trichloroacetimidates are obtained in the same manner.

The second stage consists in the catalytic nucleophilic substitution of the leaving group introduced, by paranitrophenol, by a monoterpenol or by an alcohol. Thus, after purification by chromatography on silica gel, a peracetylated β-p-nitrophenyl, β-terpenyl or β-alkyl glycoside is obtained.

Thus, the action of monoterpenols such as geraniol, nerol, α-terpineol or linalool, or of alcohols such as benzyl alcohol or 2-phenylethanol, on peracetyl-α-bromo-D-glucopyranoside or peracetyl-α-bromorutinoside is performed in the presence of silver carbonate, in an aprotic solvent such as, for example, ether, methylene chloride or chloroform, in the presence of drierite or a 0.4 nm (4 Å) molecular sieve; or in the presence of a soluble catalyst such as mercuric cyanide, in acetonitrile, in the presence of a 0.4 nm (4 Å) molecular sieve; the action of p-nitrophenol on peracetyl-α-chlororutinoside is obtained in pyridine, in the presence of silver carbonate and drierite; that of monoterpenols such as linalool, geraniol or α-terpineol on O-(peracetyl-α- and β-rutinosyl) trichloroacetimidates or O-[hexaacetyl-6-O-(α-L-arabinofuranosyl)-α- and β-D-glucopyranosyl] trichloroacetimidates is carried out in the presence of a 0.4 nm (4 Å) molecular sieve and boron trifluoride etherate or para-toluenesulphonic acid in methylene chloride or chloroform.

Chromatography of the desired peracetyl β-glycosides on silica gel is carried out by eluting with ether/petroleum ether, chloroform/ether, methylene chloride/ether or ethyl acetate/petroleum ether mixtures.

The final stage consists in the removal of the protective acetyl groups from the sugar portion of the glycosides formed; the deacetylation is performed by transesterification in methanol in the presence of a basic catalysis such as sodium methylate.

Among peracetylated saccharides which are the starting materials for these syntheses, only 1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranose is not commercially available. Its preparation may be accomplished by the synthesis described above, starting with 1,2,3,5-tetra-O-acetyl-α, β-L-arabinofuranose and 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose. However, it is preferable to carry it out by activation of the 1,2,3,5-tetra-O-acetyl-α,β-L-arabinofuranose to 3,5-di-O-acetyl-1,2-O-[(1-exo- and 1-endo-cyano)ethylidene]-β-L-arabinofuranoses using trimethylsilyl cyanide in the presence of a Lewis acid such as stannous chloride, and of the 1,2,3,4-tetra-O-acetyl-β-D-glucopyranose by tritylation to 1,2,3,4-tetra-O-acetyl-6-O-trityl-β-D-glucopyranose. The glycosylation reaction between these two synthons is performed under rigorously anhydrous conditions, in the presence of triphenylcarbonium perchlorate as a catalyst.

It is possible to apply this synthesis to the preparation of p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside, by coupling the same cyanoethylidene derivative with p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside. However, this glycosylation leads, besides the expected diholoside, to the diholoside having a 1-4 inter-saccharide bond: their separation is possible by chromatography on silica gel, eluting with an ethyl acetate/petroleum ether mixture.

EXAMPLE 1 Decaacetyl Rutin

In a 500-ml round-bottomed flask equipped with a condenser with a calcium chloride guard tube, 25 g of rutin are added with magnetic stirring to 150 ml of anhydrous pyridine. While the mixture is cooled in a cold water bath, 100 ml of acetic anhydride are then added in the course of approximately 10 minutes, and stirring is continued for 24 hours. The reaction medium is then poured into 1.5 l of icecold water: the acetylated derivative precipitates in white crystals. These crystals are filtered off, washed with water and with a little ethyl ether and then dried under vacuum in a desiccator over silica gel. 38.3 g are obtained (yield: 99%)--TLC, silica gel--ether Rf=0.22, m.p. 128-135° C.

EXAMPLE 2 2,3,4-Tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.alpha.-D-glucopyranosyl chloride

Approximately 6 g of ZnCl₂ are melted over a Bunsen burner in a crucible. It is allowed to cool under aluminium foil and 4 g of this ZnCl₂, which has been coarsely ground, are rapidly placed in a 250-ml round-bottomed flask equipped with a condenser and provided with a calcium chloride guard tube.

The following are then added with magnetic stirring:

80 ml of anhydrous chloroform, then

20 g of decaacetylated rutin;

finally, approximately 20 ml of 1,1, -dichloromethyl methyl ether are introduced in the course of 5 minutes;

the mixture is then brought to 75-77° C. for 2 hours.

The chloroform solution is decanted and the pasty residue is washed from the flask with 20 ml of chloroform, and this is combined with the chloroform solution which is evaporated under vacuum in a rotary evaporator at 35-45° C.

The oily yellow residue is taken up in 500 ml of ethyl ether which is washed with ice-cold water, with saturated Na₂ CO₃ solution and then again with ice-cold water. The organic phase is dried over Na₂ SO₄ and filtered, and the solution is concentrated in a rotary evaporator under vacuum at about 35° C. The residue, which has partially crystallized, is recrystallized in ethyl ether, and then a second time in anhydrous ethanol. The white crystals obtained are dried under vacuum in a desiccator over silica gel.

6.3 g are obtained (yield--53%)--TLC, silica gel --ether Rf=0.52, m.p. 148-150° C.

EXAMPLE 3 P-Nitrophenyl 2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside

In an Erlenmeyer equipped with a calcium chloride guard tube and a magnetic stirrer, 3 g of drierite and 1.5 g of p-nitrophenol are introduced into 50 ml of anhydrous pyridine. The mixture is stirred thus for 1 hour, and 5 g of acetochloro-α-rutinoside and 3 g of silver carbonate, freshly prepared and dried, are then added.

Stirring is continued in the dark and at room temperature for 24 hours.

The reaction medium is filtered, the precipitate is washed with a little pyridine and the filtrate is then concentrated in a rotary evaporator under vacuum at about 40-45° C. The residue is taken up twice with 25 ml of benzene and concentrated in the same manner to remove the traces of pyridine.

The residue is again taken up in 100 ml of benzene and the solution is washed with ice-cold water, N sodium hydroxide solution and then again with ice-cold water, and finally dried over Na₂ SO₄. After filtration, the filtrate is concentrated in a rotary evaporator under vacuum at 35-40° C. The reddish, pasty residue is purified by chromatography on silica gel [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70 to 230 mesh)], eluting with ethyl ether--Rf≈0.5. The purest fractions are concentrated in a rotary evaporator and the white crystals obtained are recrystallized in 95° strength ethanol. 1.4 g are thereby obtained (yield=25%)--TLC, silica gel--ether Rf=0.5, m.p. 185-187° C.

EXAMPLE 4 2,3,4-Tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.alpha.-D-alucopyranosyl bromide

1.3 g of heptaacetorutinose and 0.4 ml of acetic anhydride in 20 ml of chloroform are placed under nitrogen at -4° C. in a 50-ml round-bottomed flask, and 3.8 ml of a 33% strength solution of gaseous hydrobromic acid in acetic acid are added dropwise. Stirring is continued at -4° C. for 2 hours, and the reaction medium is then poured into 50 ml of ice-cold water. The organic phase is separated after settling has taken place, dried over anhydrous sodium sulphate and concentrated in a rotary evaporator under vacuum at 35° C. The yellow oil obtained is used without purification in the subsequent stage. However, it is possible to crystallize it by taking it up with a little ethyl ether and leaving it in the cold. On filtration under nitrogen, washing with a little ethyl ether and petroleum ether and drying in a desiccator in the cold, 310 mg of white crystals are obtained (yield--23%). M.p. 120-125° C.

EXAMPLE 5 Geranyl 2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside

In a 50-ml round-bottomed flask, the following are stirred for 24 hours under nitrogen at room temperature:

665 mg of bromo-2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-α-D-glucopyranoside (crude product of the bromination reaction);

1 ml of geraniol;

0.5 g of mercuric cyanide in 10 ml of acetonitrile.

The mixture is then concentrated in a rotary evaporator under vacuum at 35° C., and the residue is taken up in 50 ml of ethyl ether. The solid which precipitates is filtered off and rinsed with ethyl ether, and the filtrate is concentrated in a rotary evaporator under vacuum at 35° C. The oily residue is chromatographed on silica gel [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70 to 230 mesh)], eluting successively with ethyl ether/petroleum ether (10:90) to remove the excess geraniol and then with ethyl ether/petroleum ether (75:25) to elute the rutinoside. The fractions containing the rutinoside are combined and concentrated at 35° C. under vacuum in a rotary evaporator. 140 mg of a colourless paste, which it has not been possible to crystallize, are obtained (yield=19%), the product being pure in TLC--silica gel; ether/petroleum ether (3:1) Rf=0.33.

EXAMPLE 6 2,3,4-Tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamno-pyranosyl)-D-glucopyranose

In 100-ml round-bottomed flask, a mixture of 600 mg of heptaacetorutinose and 1.2 ml of benzylamine in 80 ml of ethyl ether are left with magnetic stirring and under nitrogen for 24 hours at room temperature. The reaction medium is then concentrated in a rotary evaporator under vacuum, and the oily residue is chromatographed on a column of silica gel [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70-230 mesh)], eluting with ethyl ether. The fractions containing hexaacetylrutinose are concentrated in a rotary evaporator under vacuum. 500 mg of colourless oil are obtained (yield=83%); TLC--silica gel--diethyl ether Rf=0.31.

EXAMPLE 7 O-[2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamno-pyranosyl)-.alpha.- and -β-D-glucopyranosyl]trichloroacetimidate

2.6 g of 2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-D-glucopyranose, 1.6 ml of trichloroacetonitrile and 25 ml of methylene chloride are mixed under nitrogen and at room temperature in a 100 ml round-bottomed flask. 1.6 g of anhydrous potassium carbonate are then added with magnetic stirring, and stirring is continued for 18 hours. The reaction medium is then filtered and the precipitate is rinsed with 10 ml of methylene chloride. The filtrate is concentrated in a rotary evaporator under vacuum at 35° C. and the residue is chromatographed on a column of silica [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70-230 mesh)], eluting with a 1:1 ethyl ether/methylene chloride mixture. The fractions containing the imidate are combined and concentrated in a rotary evaporator under vacuum at 35° C. 2.3 g of a colourless oil are obtained (yield=71%); TLC--silica gel--diethyl ether Rf=0.76.

EXAMPLE 8 (±)-Linalyl 2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside

In a 100-ml round-bottomed flask, a mixture of 550 mg of (±)-linalool, 650 mg of O-[2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.alpha.- and -β-D-glucopyranosyl]tri-chloroacetimidate and 1 g of 0.4 nm (4 Å) molecular sieve in 3 ml of methylene chloride are stirred under nitrogen at room temperature for 30 minutes. 22 μl of a 50% strength solution of boron trifluoride etherate in methylene chloride are then added, and are also added after 40 minutes' magnetic stirring. After a further 40 minutes' stirring, 650 mg of sodium bicarbonate are added to the reaction medium, which is then washed, volume for volume, with 0.5 M aqueous sodium bicarbonate solution and then with water. The organic phase is dried over anhydrous sodium sulphate and concentrated in a rotary evaporator under vacuum at 35° C. The oily residue is chromatographed on silica gel [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70-230 mesh)], eluting first with a 4:1 petroleum ether/diethyl ether mixture to remove the excess linalool and then with a 1:4 diethyl ether/chloroform mixture to elute the acetylated heteroside. The fractions containing the latter are combined and concentrated in a rotary evaporator under vacuum at 35° C. 190 mg of a colourless oil are obtained (yield=29%); TLC--silica gel; diethyl ether/petroleum ether (4:1) Rf=0.34.

EXAMPLE 9 Geranyl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside

In a 50-ml Erlenmeyer swept with a stream of nitrogen, 0.2 g of geranyl 2,3,4-tri-O-acetyl-6-O-(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)-.beta.-D-glucopyranoside are dissolved with mechanical stirring in 2 ml of anhydrous methanol.

0.3 ml of a sodium methylate solution (prepared from 230 mg of sodium and 100 ml of anhydrous methanol) is then added in the course of 30 seconds, and stirring is continued under nitrogen in an oil bath at 68-70° C. for 20 minutes. The Erlenmeyer is then cooled in a water/ice bath and approximately 0.2 ml of wet Dowex 50W×4(H⁺) [of a particle size corresponding to passage through a sieve of mesh aperture 74/147 μm (100/200 mesh)] is added so that the pH of the solution becomes in the region of 7. The resin is then filtered off and the filtrate is concentrated in a rotary evaporator under vacuum at 25° C. The oily residue obtained is purified by chromatography on a column of silica gel 60 [of a particle size corresponding to passage through a sieve of mesh aperture 67 μm to 199 μm (70 to 230 mesh)], eluting with a 3:1 ethyl acetate/methanol mixture. The fractions containing the geranyl β-rutinoside are combined and concentrated in a rotary evaporator under vacuum at 25° C. 110 mg of a colourless paste, which it has not been possible to crystallize, are obtained, the product being pure in TLC--silica gel; ethyl acetate/methanol (3:1) Rf=0.35.

EXAMPLE 10 2,3,4-Tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabino-furanosyl)-.alpha.- and -β-D-glucopyranoses

Gaseous ammonia is bubbled for 15 minutes into a 100-ml round-bottomed flask containing 25 ml of a 7:3 tetrahydro-furan/methanol mixture and maintained at 0° C. in crushed ice.

400 mg of 1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranoside are then added.

The flask is then allowed to return to room temperature while stirring is continued.

The reaction is followed by TLC (silica gel; methylene chloride/ether, 6:4). When there is no more starting material (after approximately 15 minutes), the reaction is stopped and the medium is concentrated to dryness under reduced pressure at 40° C.

The residue is then purified on a column of silica, eluting with a 6:4 methylene chloride/ether mixture. 250 mg of product deacetylated at the 1-position are thereby obtained (yield: 67%). TLC--silica gel--methylene chloride/ether (6:4); Rf=0.36.

EXAMPLE 11 O-[2,3,4-Tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl-.alpha. and -β-D-glucopyranosyl]trichloroacetimidate

250 mg of 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.alpha.- and -β-D-glucopyranoses, 2.5 ml of absolute methylene chloride, 0.16 ml of trichloroacetonitrile and 160 mg of anhydrous potassium carbonate are introduced into a reactor maintained under nitrogen.

The medium is left for 48 hours at room temperature with magnetic stirring. It is checked that the reaction is complete by TLC (silica gel; methylene chloride/ether, 6:4).

The medium is then taken up with 10 ml of CH₂ Cl₂ and filtered on a sinter. The organic phase is washed successively, volume for volume, with saturated aqueous NaHCO₃ solution and with ice-cold water; it is then dried over anhydrous sodium sulphate and thereafter concentrated to dryness at 35° C. under reduced pressure. 264 mg of a yellowish oil are thereby obtained (yield=88%)--TLC--silica gel--methylene chloride/diethyl ether (6:4)--Rf=0.5.

EXAMPLE 12 Geranyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-arabinofuranosyl)-.beta.-D-glucopyranoside

250 mg of O-[2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-α- and -β-D-glucopyranosyl] trichloroacetimidates, 250 μl of geraniol and 1.5 ml of anhydrous methylene chloride are introduced into a 50-ml reactor maintained under nitrogen.

The medium is maintained under nitrogen and with magnetic stirring; 20 μl of a 50% strength solution of boron trifluoride etherate in methylene chloride are added dropwise.

After 3 hours' reaction, it is checked that the reaction is complete by TLC (silica gel, methylene chloride/ether, 7:3), and 50 mg of sodium bicarbonate are then added to the medium. 10 ml of methylene chloride are added and the reaction medium is washed, volume for volume, with ice-cold 0.5 M sodium bicarbonate solution and then with ice-cold water. The organic phase is dried over anhydrous sodium sulphate and taken to dryness at 35° C. under reduced pressure. The oily residue is rapidly purified on a column of silica, eluting first the excess geraniol with a 1:1 ether/petroleum ether mixture, and then the product with an 8:2 methylene chloride/ether mixture. 150 mg of a colourless oil are thereby obtained (yield =58%). TLC: silica gel, methylene chloride/ether (8:2), Rf=0.49.

EXAMPLE 13 p-Nitrophenyl 2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside

In a 100-ml round-bottomed flask, 1.42 g of p-nitrophenyl β-D-glucopyranoside and 1.5 g of anhydrous trityl chloride are introduced into 10 ml of anhydrous pyridine, and the flask is left in the dark with stirring at 40° C. The reaction is followed by TLC, adding trityl chloride if necessary. After 24 hours, the flask is brought to room temperature and 6 ml of acetic anhydride are added. The reaction medium is left for 48 hours with stirring, the reaction being followed by TLC (silica gel, ether/petroleum ether, 7:3): it is taken up in 500 ml of ice-cold water and stirred for 2 hours. The mixture is then filtered through celite and the celite is rinsed with dichloromethane. The organic phase is washed successively with ice-cold water, 10% strength hydrochloric acid, saturated aqueous sodium bicarbonate solution and then ice-cold water. It is dried over sodium sulphate and concentrated under vacuum at 35° C. The residue is chromatographed on a column of silica, eluting with a 7:3 ether/petroleum ether mixture. The fractions containing the heteroside are combined and concentrated under vacuum at 35° C. 2.6 g of an oil are obtained (yield=83%), the product being pure in TLC: silica gel, ether/petroleum ether (7:3), Rf=0.33.

EXAMPLE 14 1,2,3,5-Tetra-O-acetyl-α,β-L-arabinofuranose

A mixture of anhydrous L-arabinose (10 g) and anhydrous methanol (200 ml) is treated with 1.06 M methanolic hydrochloric acid [prepared by adding acetyl chloride (4.7 ml) and anhydrous methanol (63 ml) at 0° C.]; the mixture is stirred overnight at 0-5° C. Pyridine (40 ml) is added to neutralize the mixture, which is then concentrated. The residue is taken up several times in pyridine, which is removed by distillation, and it is then dissolved in 80 ml of pyridine. Acetic anhydride (30 ml) is added in the cold state, and the solution is left at room temperature for 2 days. Extraction of the reaction medium with ethyl acetate or dichloromethane gives a syrupy product which is then dissolved in a mixture of acetic acid (100 ml) and acetic anhydride (25 ml); 5 ml of concentrated sulphuric acid are added at 0° C., and the mixture is left overnight at room temperature. The solution is then immersed in crushed ice (150 g) and the mixture is stirred for 2 hours and then extracted with chloroform. The extract is washed with water and then with aqueous sodium bicarbonate solution; the residue obtained after concentration of the organic phase is chromatographed on a column of silica gel (eluant: benzene/ether gradient) and gives 1,2,3,5,-tetra-O-acetyl-α,β-L-arabinofuranose in the form of a syrup (18 g, 85%), Rf=0.46 (eluant: benzene).

EXAMPLE 15 3,5-Di-O-acetyl-1,2-O-[1-exo- and 1-endo-cyano)ethylidene]-β-L-arabinofuranose

Anhydrous stannous chloride (360 mg) and trimethylsilyl cyanide (3 ml) are added to a solution of 1,2,3,5-tetra-O-acetyl-α,β-L-arabinofuranose (3 g) in acetonitrile (10 ml). The mixture is stirred overnight at room temperature, then diluted with ether and washed with aqueous sodium bicarbonate solution (3×75 ml) and then with water. The organic phase is concentrated and the residue is chromatographed on a column (eluant: benzene/ether gradient) to give the 1-exo-cyano (994 mg; 37%) and 1-endo-cyano (700 mg; 26%) product. Crystallization with ether/pentane gives the 1-exo-cyano isomer (35%), m.p. 66-69° C., [α]_(D) -6° (C1), Rf=0.56 (eluant: benzene/ether, 3:2). Crystallization with toluene gives the 1-endo-cyano isomer (23%), m.p. 107-110° C., [α]_(D) +51° (C1), Rf=0.37.

EXAMPLE 16 1,2,3,4-Tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranose and p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside

Into two round-bottomed flasks closed and joined by a tube shaped like a tuning-fork, there are introduced, on the one hand (in one flask) a solution of tritylated glucoside (0.55 mmol) and 3,5-di-O-acetyl-1,2-O-[(1-exo- and 1-endo-cyano)ethylidene]-β-L-arabinofuranose (0.5 mmol) in nitromethane (2 ml), and on the other hand (in the other flask) a solution of triphenylcarbonium perchlorate (0.05 mmol) in 0.2 ml of nitromethane. Both solutions are lyophilized, and then 2 ml of distilled benzene are introduced into each flask, the contents being lyophilized again; the operation is repeated a second time; the flasks and the reactants are thus dried for several hours. Dichloromethane (2 ml) is distilled in situ into each of the two flasks. The two solutions are then combined and left to react overnight at room temperature and in the dark [the lyophilization and also the drying of the reactants, as well as the distillation of a benzene and dichloromethane over CaH₂, are performed at a pressure of 0.533 Pa (4×10⁻³ mmHg)]. The brilliant yellow reaction medium is treated with 1 ml of pyridine/water (3:1), and the decolorized solution is then diluted with chloroform (50 ml), washed with water (3×30 ml) and concentrated. The residue is purified on a column of silica gel, eluting with benzene/ether or ethyl acetate/petroleum ether gradient, to yield:

from 1,2,3,4-tetra-O-acetyl-6-O-trityl-β-D-glucopyranose, 1,2,3,4-tetra-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-β-D-glucopyranose, m.p. 106.5-108.5° C. (ether/pentane); TLC: silica gel; benzene/ether (3:2), Rf=0.35.

from p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-trityl-β-D-glucopyranoside, p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside; TLC: silica gel; ethyl acetate/petroleum ether 1:1, Rf=0.24 and p-nitrophenyl 2,3,6-tri-O-acetyl-4-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside; TLC, silica gel; ethyl acetate/petroleum ether (1:1), Rf=0.28.

EXAMPLE 17 Geranyl 6-O-(α-L-arabinofuranosyl)-β-D-gluconyranoside

200 mg of geranyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside are dissolved under nitrogen in 5 ml of anhydrous methanol with magnetic stirring. 0.1 ml of a methanolic solution of sodium methylate (prepared from 20 mg of sodium and 10 ml of methanol) is added. Stirring is continued for 4 hours at room temperature, and the solution is then neutralized by adding Dowex 50W×4 (H⁺) resin.

The mixture is filtered and the solution is concentrated to dryness in a rotary evaporator. The oily residue is purified by chromatography on a column of silica gel, eluting with an 8:2 chloroform/methanol mixture to yield 105 mg of geranyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside in the form of a syrup. Yield 81%, TLC: silica gel, chloroform/methanol (8:2), Rf=0.17.

EXAMPLE 18 p-Nitrophenyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside

By deacetylation of p-nitrophenyl 2,3,4-tri-O-acetyl-6-O-(2,3,5-tri-O-acetyl-α-L-arabinofuranosyl)-.beta.-D-glucopyranoside according to the above procedure, p-nitrophenyl 6-O-(α-L-arabinofuranosyl)-β-D-glucopyranoside is obtained. TLC: silica gel, ethyl acetate/isopropanol/water (65:30:10), Rf=0.69.

EXAMPLE 19 p-Nitrophenyl 1-β-D-apiose

2 g (6.28 mmol) of 1,2,3,5-tetra-O-acetyl(3')-β-D-apiose and 4 g (28.8 mmol) of paranitrophenol in a 100 ml round-bottomed flask are dried by coevaporation in toluene.

Then 40 mg (0.2 mmol) paratoluene sulfonic acid are added and the mixture is heated at 95° C. for 50 minutes under vacuum 10⁻² mm Hg). After cooling, the reactional medium is dissolved in 200 ml CH₂ Cl₂ and several times washed with a saturated solution K₂ CO₃.

The oily residue is dried, concentrated then purified by chromatography on silica gel 60 column (230-240 Mesh), eluting successively with ethyl ether/petroleum ether (50/50).

1.4 g of 2,3,5-tri-O-acetyl(3')-O-paranitrophenyl-1-β-D-apiose. M.p. 155-156° C. (yield: 56%) and

0.2 g of 2,3,5-tri-O-acetyl(3')-O-paranitrophenyl-1-a-D-apiose (yield: 8%) are obtained.

1.0 of 2,3,5-tri-O-acetyl(3')-O-paranitrohenyl-1-β-D-apiose has been deacetylated by sodium methylate in anhydrous methanol.

After purification, 0,65 g paranitrophenyl-1-β-D-apiose (yield: 95%) are obtained.

Ariofuranosylglucosides

The general method used to get terpenols and alcohols apiofuranosylglucosides is the same that the previous one described for the rhamnosylglucosides and arabinosylglucosides synthesis.

It is possible to apply this synthesis to the preparation of 1,2,3,4-tetra-O-acetyl-O-(2,3,5-tri-O-acetyl(3')-6β-D-apiofuranosyl)-6-β-D-glucopyranose by coupling the 3,5-di-O-acetyl(3')-(endo or exo-cyanoethylidene)-1,2-β-D-apiofuranose and the 1,2,3,4-tetra-O-trityl-6-β-D-glucopyranose.

The diholoside heptaacetate is released from the anomeric position of the glucose, transformed in its trichloroacetimidate derivate which is the starting product for respective condensations with:

benzylic alcohol, phenylethylic alcohol, geraniol, nerol,

(R)(S)-a-terpineol, (R)-a-terpineol, (R)(S)-β-citronellol,

(S)-citronellol, (R)(S)-linalool, (S)-linalool.

EXAMPLE

50 mg (0.7 mmol) trichloroacetimidate-1-(2,3,4-tri-O-acetyl-O-(2,3,5-tri-O-acetyl(3')-.beta.-D-apiofuranosyl)-6-O-(aβ)-D-glucopyranose) and 38 ml (8.6 mmol) of (R)(S)-linalool are dissolved in 2 ml anhydrous CH₂ Cl₂ and a catalytic quantity of BF₃ :OEt₂ is added. The reactional medium is neutralized and extracted by classical methods. After purification and action of sodium methylate, the linalyl diholoside is recovered with a yield of about 50%.

B. MEASUREMENT OF THE ENZYMATIC ACTIVITIES AND PURIFICATION OF THE ENZYMES

1--Measurement of the Enzymatic Activities

The glycosidase activities are determined by incubating 0.1 ml of 4 mM substrate (Glc-pNP, Ara-pNP, Api-pNP, Rha-pNP) in a 100 mM acetate buffer (pH 4.2) with 0.1 ml of enzyme solution at 40° C. for 20 minutes. The liberation of pNP is estimated by adding 0.6 ml of 1 M sodium carbonate to the incubation medium and then measuring the optical density at 400 nm. 1 nkat of activity corresponds to the liberation of 1 nmol of pNP per second.

Two glycosidases, α-arabinosidase and α-rhamnosidase, were isolated and purified from commercial preparations containing complex mixtures of enzymes.

Sweet almond β-glucosidase (Koch-Light, Great Britain, Batch No. 2872-01), not displaying any α-arabinosidase, β-apiosidase and α-rhamnosidase type contaminant activity (24 h of incubation at 40° C., pH 4.2), was used as it is.

The β-apiosidase present in a commercial enzyme preparation (Klerzyme 200, Gist-brocades, France) was used without further purification.

2--Purification of the Enzymes

2-1. Purification of an α-L-rhamnopyranosidase

The α-rhamnosidase was purified from naringinase (Sigma) rich in these activities. The β-glucosidase activities, although low (0.1% of the α-rhamnosidase activity), but capable of liberating glucose over long incubation periods, were removed by the technique of chromatofocusing.

The Experimental Protocol:

Purification of α-rhamnosidase by chromatofocusing of naringinase on PBE 94 gel

50 mg of naringinase, taken up in 4 ml of 25 mM imidazole buffer (pH 7.4), are dialysed against the same buffer (25 ml) overnight (5° C.). The dialysate is injected onto a column (1.0×40 cm) of ion exchanger (Polybuffer exchanger PBE 94, Pharmacia, Sweden) equilibrated with the same buffer. The proteins bound to this gel are eluted by a pH gradient from 7.4 to 3.7, created during the migration of Ampholines [Polybuffer 74 (Pharmacia) adjusted beforehand to pH 3.7] at a flow rate of 44 ml/h.

After the gradient is completed, the passage of 1 M NaCl in 100 mM acetate buffer (pH 3.7) desorbs the proteins retained.

2.8-ml fractions are collected, on which the α-rhamnosidase and β-glucosidase activities, the pH and the optical density at 280 nm are measured.

The chromatographic profile is shown in FIG. 1, to which the legend is as follows:

    ______________________________________                                                  Rhamnosidase activity d. α                                        □--□β -Glucosidase activity                         ◯--◯ Absorbance at 280 nm                              +--+ pH gradient                                                             ______________________________________                                    

The α-rhamnosidase activities are eluted in the pH gradient, whereas the whole of the β-glucosidase activity is retained up to pH 3.7 (pI<3.7) and eluted by the passage of a 1M NaCl solution.

This technique shows that naringinase contains at least 3 (rhamnosidase) isoenzymes of isoelectric points pI 6.2, pI 5.7 and pI<3.7. The second (317 nkat/ml), showing no residual β-glucosidase activity even after more than 24 hours' incubation at 40° C., is chosen for the tests of enzymatic hydrolysis of natural and synthetic glycosides.

The overall recovery yield of the α-rhamnosidase activities is approximately 62%.

2-2. Purification of an α-L-arabinofuranosidase

Among the commercial enzyme preparations studied, Hemicellulase REG-2 (Gist Brocades, France) proved rich in α-arabinosidase activity. Nevertheless, it displays substantial activities of the β-glucosidase and α-rhamnosidase type. For example, 250 mg of Hemicellulase contain 2,327 nkat of α-arabinosidase activity, 2,566 nkat of β-glucosidase activity and 236 nkat of α-rhamnosidase activity. Several chromatographic techniques are applied successively in order to isolate and purify the α-arabinosidase.

Molecular sieving of Hemicellulase on Ultrogel AcA 44 Fractionation

The enzyme solution (250 mg of Hemicellulase in 3 ml of 100 mM citrate-phosphate buffer, pH 7.2) is dialysed against the same buffer (500 ml) overnight (+5° C.). The dialysate is then placed on a column (1.6×100 cm) of Ultrogel AcA 44 (IBF, France) equilibrated beforehand with the same buffer. The column is then eluted with the above buffer at a flow rate of 9 ml/h. 1.2-ml fractions are collected and the α-arabinosidase, β-glucosidase and α-rhamnosidase activities, as well as the optical density at 280 nm, are measured.

Results

The chromatographic profile is shown in FIG. 2, the legend to which is as follows:

    ______________________________________                                                  Arabinosidase activity -solid. α                                  □--□ β-Glucosidase activity                         ▪--▪ α-Rhamnosidase activity                         ◯--◯ Absorbance at 280 nm.                           ______________________________________                                    

Molecular sieving on Ultrogel AcA 44 enables the two major α-arabinosidase and β-glucosidase activities to be separated. The α-rhamnosidase activities are co-eluted with the α-arabinosidase activities. The majority of the proteins present in the initial enzyme solution is eluted with the α-arabinosidase activities.

The fraction (52 ml) corresponding to the α-arabinosidase activities (1,750 nkat) also contains trace activities, that is to say β-glucosidase (23.4 nkat) and α-rhamnosidase (37.4 nkat), equivalent to 1.3% (β-glucosidase) and 2.1% (α-rhamnosidase) relative to the α-arabinosidase activity.

Ion Exchange Chromatography on DEAE-Sepharose CL-6B of the α-arabinosidase Fraction Derived from the Molecular sieving Fractionation

The α-arabinosidase-rich fraction (52 ml) was dialysed against 500 ml of 25 mM imidazole-HCl buffer (pH 7.5) overnight (+5° C.). The dialysate is then placed on a column (1.6×40 cm) of DEAE-Sepharose CL-6B (Pharmacia) equilibrated beforehand with the same buffer. The gel is first washed with this buffer at a flow rate of 108 ml/h. The proteins retained on the column are then eluted by a linear gradient of sodium chloride (from 0 to 0.4 M) in the same buffer (imidazole-HCl) at a flow rate of 40 ml/h. 4-ml fractions are collected, on which the α-arabinosidase, β-glucosidase and α-rhamnosidase activities and the optical density at 280 nm are measured.

Results

The chromatographic profile is shown in FIG. 3, the legend to which is as follows:

    ______________________________________                                                 Arabinosidase activity -solid. α                                   □--□ β-Glucosidase activity                         ▪--▪ α-Rhamnosidase activity                         ◯--◯ Absorbance at 280 nm                             --- Ionic strength gradient (NaCl)                                            ______________________________________                                    

Under these conditions, the three activities are well separated. The peak eluted (69 ml) using 0.3 M NaCl and corresponding to the α-arabinosidase activities (365 nkat) now contains only low β-glucosidase (0.43 nkat) and α-rhamnosidase (0.32 nkat) activities, equivalent to 0.1% (β-glucosidase) and 0.08% (α-rhamnosidase) relative to the α-arabinosidase activity.

It should be noted that, on completion of this stage, the α-arabinosidase fraction contains much less protein than the initial enzyme solution.

This stage enables the α-arabinosidase activity to be purified approximately 14-fold. However, since the presence of low β-glucosidase and α-rhamnosidase activities can interfer in the hydrolysis of natural and synthetic glycosides, the purification was refined by the application of a further (affinity) chromatographic technique.

Affinity Chromatography on Concanavalin A-Ultroael of the α-arabinosidase Fraction Derived from the Chromatographic Stage on DEAE-Sepharose CL-6B Fractionation

The α-arabinosidase fraction (69 ml) is concentrated in a dialysis sack covered with Sephadex G-200 gel (Pharmacia) to 12 ml. It is dialysed first against a 50 mM Tris-HCl buffer (pH 7.2) containing 0.1 M NaCl and 0.1 mM MnCl₂ overnight (+5° C.), and then injected onto a concanavalin A-UG gel (IBF, France) (1.0×10 cm), equilibrated beforehand with the above buffer, at a flow rate of 30 ml/h. The column is eluted with methyl α-D-mannopyranoside (Serva, FRG) in the same buffer, first with a linear gradient (0→>0.15 M) and then isocratically (0.15 M). 1.5-ml fractions are collected, on which the optical density and the α-arabinosidase, β-glucosidase and α-rhamnosidase activities are measured. The combined fractions displaying α-arabinosidase activities are dialysed against 100 mM acetate buffer (pH 4.2) over-night (+4° C.) to remove the methyl α-D-mannopyranoside.

Results

The chromatographic profile is shown in FIG. 4, the legend to which is as follows:

    ______________________________________                                         Arabinosidase activity -solid. α                                           ◯--◯ Absorbance at 280 nm                              ---- Methyl α-D-mannopyranoside gradient                               ______________________________________                                    

This stage has made it possible to remove a majority of the proteins and all the residual β-glucosidase and α-rhamnosidase activities, verified after concentration of the fractions by dialysis against dry Sephadex G-200 gel. The major α-arabinosidase peak (17 ml; 75 nkat) represents 20.5% of the initial activity injected (365 nkat). A part of the activity (62.5 nkat) trails on elution with methyl α-D-mannopyranoside and corresponds to 17.1% of the initial activity. The enzyme was strongly bound to the gel, and the methyl α-D-mannopyranoside enabled only 37.6% of the initial activity to be eluted (similar results are observed for other enzymes).

As a result of this final stage, the α-arabinosidase activity has been purified approximately 27-fold. The enzyme solution used in the hydrolysis tests contains 29.9 nkat/ml of activity.

The numerical results corresponding to the different stages during the purification of α-arabinosidase from Hemicellulase are collated in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Purification of α-arabinosidase from Hemicellulase REG-2                             Total                                                                              Activity                                                                           Total                                                                               Protein                                                                            Specific                                             Volume activity yield proteins(1) yield activity Purification                 Stage (ml) (nkat) (%) (mg) (%) (nkat mg                                                                         .sup.-1) factor                             __________________________________________________________________________     Crude Hemi-                                                                            3.2 2327                                                                               100 322.6                                                                               100 7.2   1                                             cellulase                                                                      (250 mg)                                                                       Sieving 52 1759 75.6 112.3 34.8  15.7 2.2                                      on Ultro-                                                                      gel AcA 44                                                                     Ion exchange 69  365 15.7 3.6 1.1 101.3 14.1                                   on DEAE-                                                                       Sepharose                                                                      CL-6B                                                                          Affinity on 34  75  3.2 0.4 0.1 192.3 26.1                                     Concanavalin                                                                   A-Ultrogel                                                                   __________________________________________________________________________      (1)Proteins are assayed according to Lowry et al. (1951) "Protein              measurement with the Folin phenol reagent", J. Biochem, 193, pages 265-27

Properties of the Purified α-arabinosidase pH Optimum

The enzyme solution is incubated in the presence of its substrate (Ara-pNP) in universal buffer (Britton and Robinson type) of pH varying from 3.0 to 7.0. The activities measurements are carried out at the various incubation pH values under the conditions described at the beginning of this section A. The residual activity as a function of the pH is shown in FIG. 5a (curved ).

The α-arabinosidase activity is maximal at around pH 3.7-4.0.

Stability of the Activity as a Function of the pH

The enzyme solution is incubated for 50 min at 60° C. in a universal buffer whose pH varies from 3.0 to 6.5. The samples are then dialysed against 1 l of 100 mM acetate buffer (pH 4.2) for 5 hours (+5° C.). The residual activity measured is shown in FIG. 5a (curver ).

The α-arabinosidase activity is relatively stable between pH 3.8 and 4.9. This stability decreases rapidly at pH values below 3.5 and above 5.5.

Temperature Optimum

The α-arabinosidase activity is determined after incubation of the reaction medium at different temperatures varying from 5° C. to 80° C. The relative activity as a function of the incubation temperature is shown in FIG. 5b (curve ).

The activity is maximal at 60° C.

Thermal Stability

The enzyme solution is maintained at different temperatures (from 5° C. to 80° C.) in 100 mM acetate buffer (pH 4.2) for 30 minutes: the residual activity is then measured and the results are shown in FIG. 5b (curve ).

The α-arabinosidase activity is stable up to 60° C., and above this the stability decreases abruptly. It is almost totally inactivated after a treatment at 70° C. for 30 minutes.

C. ENZYMATIC HYDROLYSES

The separate or sequential action of four enzymes, an α-L-arabinofuranosidase (E.C.3.2.1.55, designated α-arabinosidase), an α-L-rhamnopyranosidase (E.C.3.2.1.40, designated α-rhamnosidase), a β-D-apiofuranosidase (designated β-apiosidase) and a β-D-glucopyranosidase (E.C.3.2.1.21, designated β-glucosidase) was studied on different glycosidic substrates. The latter are, on the one hand p-nitrophenyl or geranyl α-L-rhamnopyranosyl-(1→>6)-β-D-glucopyranosides (designated Rha-Glc-pNP or Rha-Glc-Ger), geranyl β-D-apiofuranosyl-(1→>6)-β-D-glucopyranoside (designated Api-Glc-Ger) and p-nitrophenyl α-L-arabinofuranosyl-(1→>6)-β-D-glucopyranoside (designated Ara-Glc-pNP), and on the other hand a glycosidic extract purified from a muscat must.

It should be noted that the four synthetic glycosides studied possess the same structures in respect of their carbohydrate portion as the grape terpene glycosides, but differ in their aglycone which, for three of them, is p-nitrophenol (pNP).

The enzymatic hydrolysis is followed by thin-layer chromatography (TLC) and by gas chromatography (GC).

Thin-layer Chromatography (TLC)

Thin-layer adsorption chromatography was carried out on pieces of aluminium foil covered with a thin layer (0.2 mm) of silica gel (5553 Silica gel 60 without a fluorescence indicator, Merck). An ethyl acetate/isopropanol/water (65:30:10 v/v/v) mixture was used as a migration solvent.

The sugars and the glycosides are visualized by means of the following mixture, prepared immediately before use: 0.2% naphthoresorcinol in ethanol/concentrated sulphuric acid (19:1 v/v). The application of this visualizing agent is followed by drying the plates in the oven (105° C.) for 15 minutes. Apart from their migration distance, the various compounds are also distinguished by their colour. The glycosides are purple-red, the arabinosides are bluish, the apiosides are green, rhamnose greenish pink, arabinose blue, apiose green and glucose pink.

Moreover, TLC enabled the portion of the glycosidic extract not hydrolysed by the enzymes to be recovered. The zone of the chromatographic plate where the corresponding spot is localized was scrapped off, the silica gel recovered and suspended in 50 ml of methanol. After being left overnight with gentle stirring, the suspension is filtered on a Buchner funnel and the gel is washed with 3×10 ml of methanol. The organic eluates are combined, taken to dryness under vacuum at 40° C. and then taken up in 500 μl of ultrapure water. The aqueous sample thereby obtained is then ready for acid hydrolysis.

Gas Chromatography (GC)

This technique was used in order to analyse both the sugars and the terpene glycosides and also the free terpenols. For this purpose, two different chromatographic systems were employed.

GC of the Glycosides

Glycosides and sugars are non-volatile compounds, and are hence not suited, as such, to an analysis by GC. It is essential to convert them to volatile compounds by means of a selected reagent. Trimethylsilyl derivatives were hence formed, according to the following protocol:

40 μl of glycosidic extract and 60 μl of a solution of Glc-pNP (internal standard) at a concentration of 50 mg/l in ethyl acetate are introduced into a suitable flask. The mixture is taken to dryness at 40° C. with a stream of nitrogen. 40 μl of the silylating reagent (Tri-sil, Pierce, Rockford, Ill., USA) are then introduced; the flask is sealed and maintained at 40° C. for 20 minutes. After rapid cooling, the silylated sample is then ready for analysis by GC.

The apparatus used consists of a Series 30C gas chromatograph, an "on-column" injector (injected volume 0.5 μl) and a flame ionization detector (Girdel, France). A thin film (0.20 μm) of an OV-1 (Girdel) apolar silicone phase is grafted onto the inner wall of the capillary column (50×0.32 mm I.D.). The oven temperature is programmed from 125 to 305° C. at the rate of 5° C./minute, then maintained at 305° C. for 15 minutes. Finally, the detector temperature is set at 300° C. and hydrogen is used as the carrier gas at a pressure of 120 kPa.

GC of the Terpenols

The monoterpene alcohols studied here are sufficiently volatile to be chromatographed as they are. To the pentane extract containing them, the internal standard, 4-nonanol (for synthesis, Merck, Darmstadt, FRG) is added in the proportion of 1 μl of a solution at a concentration of 2.89 mg/ml in pentane per 50 μl of medium. The mixture is then dried over sodium sulphate, filtered through glass wool and then concentrated to a volume in the region of 100 μl. For this purpose, the pentane is first removed using a conventional still, and then a Dufton type column of a size suited to the volume remaining to be concentrated.

The constituents of the concentrated extract are separated using a capillary; column (25 m×0.32 mm I.D.) containing a CP wax 52 CB (Chrompack, Middelburg, Netherlands) polar phase. The grafted polyethylene glycol film was selected thick (1.28 μm) in order to permit the injection of large sample volumes, up to 4 μl. The analysis is performed using a Fractovap Series 2900 (Carlo Erba, Milan, Italy) chromatograph, equipped with a flame ionization detector maintained at 250° C. and an "on-column" injector. Hydrogen was used as the carrier gas at a pressure of 60 kPa, and the oven temperature was programmed as follows: isothermal at 70° C. for 5 minutes, followed by a rise to 195° C. at the rate of 2° C./minute and a plateau at 195° C. for 15 minutes.

High Performance Liquid Chromatography (HPLC)

The benefits of high performance liquid chromatography were exploited for isolating some terpene glycosides. For this purpose, fractions were collected as they emerged from the chromatographic system (more especially on elution of the peaks A and B) during the separation of the constituents of the glycosidic extract. (FIG. 10 below). Fifteen successive injections of 20 μl each enabled sufficient material to be isolated for the enzymatic hydrolyses and the analyses by GC to be performed in order to identify the compounds in question.

The chromatographic system consists of the following components: a Vista 5500 chromatograph equipped with a variable wavelength UV/visible spectrophotometer (Varian Assoc., Sunnyvale, Calif., USA); a six-way injection valve (Valco) equipped with a 20 -μl loop; a stainless steel column (220×4 mm I.D.) filled with Spheri-5 (Brownlee Labs., Santa Clara, Calif., USA) octadecyl grafted silica of small particle size (5 μm); as well as a precolumn (37×4 mm I.D.) packed with the same stationary phase.

The chromatography is carried out using a water/+acetonitrile aqueous-organic mobile phase (reversed-phase polarity chromatography) and an elution graduated according to an increase in the acetonitrile content from 30 to 40% (by volume) in the course of 10 minutes. The elution solvent is pumped at a flow rate of 1 ml/min. The detection is performed at 200 nm and 0.5 absorbance unit full-scale.

1--ENZYMATIC HYDROLYSES ON SYNTHETIC SUBSTRATES

In all the tests relating to the enzymatic hydrolysis of the synthetic substrates, the same glycosidase activity (0.15 nkat) is used for each of the enzymes (α-arabinosidase, β-apiosidase, α-rhamnosidase, β-glucosidase).

The hydrolysis and analysis protocols are given in plan form (Table 2).

The main abbreviations used will be recalled here:

    ______________________________________                                         pNP         p-nitrophenol                                                        Ara-pNP p-nitrophenyl α-L-arabinofuranoside                              Rha-pNP p-nitrophenyl α-L-rhamnopyranoside                               Api-pNP p-nitrophenyl β-D-apiofuranoside                                  Rha-Glc-pNP p-nitrophenyl α-L-rhamnopyranosyl-(1→6)-β-        D-glucopyranoside                                                             Rha-Glc-Ger geranyl α-L-rhamnopyranosyl-(1→6)-β-D-                       glucopyranoside                                                    Ara-Glc-pNP p-nitrophenyl α-L-arabinofuranosyl-(1→6)-β                 -                                                                     D-glucopyranoside                                                             Ara-Glc-Ner neryl α-L-arabinofuranosyl-(1→6)-β-D-                        glucopyranoside                                                    Ara-Glc-Ger geranyl α-L-arabinofuranosyl-(1→6)-β-D-                      glucopyranoside                                                    Api-Glc-Ger geranyl β-D-apiofuranosyl-(1→6)-β-D-                          glucopyranoside                                                    Glc-pNP p-nitrophenyl β-D-glucopyranoside                                 Glc-Lin linalyl β-D-glucopyranoside                                       Glc-Ner neryl β-D-glucopyranoside                                         Glc-Ger geranyl β-D-glucopyranoside                                     ______________________________________                                    

                                      TABLE 2                                      __________________________________________________________________________     Plan of the protocol for enzymatic hydrolysis of the synthetic substrates*               1st Stage                                                                              2nd Stage                                                    __________________________________________________________________________               Addition of         Addition of 50 μl of a solution of                                            either 50 μl of  β-glucosidase                                       (0.15 nkat) to TEST I                               a solution of  Reincubation 40° C., 90 mins, then                      200 μl of 4 mM α-arabinosidase  15 μl → TLC                 Ara-Glc-pNP in (0.15 nkat) Incubation 40° C., 90 min 36 μl                                       → pNP liberated                             100 mM acetate (TEST I) then                                                   buffer (pH 4.2)  12 μl → TLC                                          or 50 μl of a 30 μl → pNP liberated                               solution of                                                                    β-glucosidase                                                             (0.15 nkat)                                                                    (TEST II)                                                                      Addition of  Addition of 50 μl of a solution of                             either 50 μl of  β-glucosidase (0.15 nkat) to TEST III                                              a solution of  Reincubation 40°                                       C., 90 min, then                                   200 μl of α-rhamnosidase  extraction with pentane                     Rha-Glc-pNP (4 mM) (0.15 nkat) Incubation 40° C., 90 min (5                                         × 250 μl) - GC                            or of (TEST III) then 15 μl - TLC                                           Rha-Glc-Ger  extraction with pentane 36 μl - pNP liberated                  (solution not or 50 μl of (5 × 250 μl) → GC                 titrated) in 100 mM a solution of 12 μl → TLC                        acetate buffer β-glucosidase 30 μl → pNP liberated                                           (pH 4.2) (TEST IV)                              __________________________________________________________________________      *The controls are carried out under the same conditions, in the presence       of the inactivated enzymes (95° C., 30 minutes)                   

The results obtained in TLC are shown in FIG. 6, to which the legend is as follows:

Monitoring by TLC of the Enzymatic Hydrolysis of Ara-Glc-PNP (a), Rha-Glc-vNP (b) and Api-Glc-Ger

1a. Ara-Glc-pNP+β-glucosidase

2a. Ara-Glc-pNP+α-arabinosidase

3a. Ara-Glc-pNP+α-arabinosidase+β-glucosidase

1b. Rha-Glc-pNP+β-glucosidase

2b. Rha-Glc-pNP+α-rhamnosidase

3b. Rha-Glc-pNP+α-rhamnosidase+β-glucosidase

The action of β-glucosidase on the different synthetic substrates gives rise to no modification.

The action of the α-arabinosidase on Ara-Glc-pNP causes the disappearance of this substrate and the appearance of arabinose and Glc-pNP.

Similarly, the action of α-rhamnosidase on Rha-Glc-pNP or Rha-Glc-Ger gives rise to the disappearance of these substrates and the appearance of rhamnose and Glc-pNP or Glc-Ger.

This constitutes the first stage of the hydrolysis.

The consecutive action of β-glucosidase on each of these media previously incubated with α-arabinosidase or α-rhamnosidase leads to the disappearance of Glc-Ger and Glc-pNP and the appearance of glucose (second stage).

As regards the corresponding aglycones (pNP or geraniol), they are liberated only after the sequential action of both glycosidases: α-rhamnosidase or α-arabinosidase, and then β-glucosidase.

In another aspect, the action of the β-apiosidase (present in Klerzyme 200) on Api-Glc-Ger leads to the disappearance of the substrate and the appearance of apiose and glucose.

These results show that the enzymatic hydrolysis of the glycosides studied proceeds well with the two stages described above.

2--ENZYMATIC HYDROLYSIS OF A GLYCOSIDIC EXTRACT PROTOCOL FOR PRODUCTION OF THE GLYCOSIDIC EXTRACT

The glycosidic extract was obtained by extraction of a must of Muscat de Frontignan variety grapes collected at maturity in the vines of the Station Exprimentale de Pech Rouge (INRA Gruissan, France), followed by removal of the free terpenols and sugars.

In view of the large number of experiments to be carried out, it was essential to have recourse to a supply of glycoside which was consistent. To this end, the procedure was performed on a large volume (80 liters) of previously centrifuged and sulphite-treated (50 ppm) must, and the extraction of the glycosidic material was carried out using 80 grams of active charcoal traditionally used in oenology (charcoal type CXV; Ceca S.A., Velizy-Villacoublay, France). The mixture was kept stirred for 4 hours and then left standing overnight. The charcoal was then recovered by filtration through cellulose filters (porosity 40-50 μm).

In order to remove the majority of the sugars also extracted by the charcoal, the latter was washed with 4×150 ml of water and filtered on a Buchner funnel. Monitoring by TLC enabled it to be verified that the sugar content decreased at each stage without elution of the glycosides taking place. The latter were then recovered by washing with 5×200 ml of acetone. Here too, monitoring by TLC enabled the quantitative elution of the glycosidic material to be checked. The acetone was removed by evaporation under vacuum and the sample was taken up in 40 ml of ultrapure water. A more exhaustive purification of the glycosidic extract was then carried out by fractionation on an Amberlite XAD-2 (Rohm & Haas Co., Philadelphie, Calif., USA) organic resin prepared according to the following protocol: grinding and sieving of the particles of size corresponding to a sieve of mesh aperture 175-350 μm (between 80 and 40 mesh), followed by three washes in a Soxhlet with methanol, acetonitrile and diethyl ether, in that order, for 8 hours for each solvent. The fractionation process, developed at the Laboratoire des Aromes et des Substances Naturelles (Aromas and Natural Substances Laboratory), has already been described in detail. In this study, it was applied twice consecutively, and comprised the following stages:

A resin, suspended in methanol, is poured into a glass column (35×1 cm) terminating in a Teflon tap and a glass wool plug. After settling, the resin layer measures approximately 20 cm. Several 50-ml portions of methanol are then passed through, followed by washing with 50 ml of diethyl ether and finally equilibration of the resin using 100 ml of ultrapure water. The column is then ready for use.

The 40 ml of glycosidic extract are passed through the column at a flow rate of between 2 and 2.5 ml/minute. The column is then washed with 100 ml of water to remove residual sugars, and with 100 ml of pentane in order to elute the free terpenols bound by the resin, the flow rate being the same as above.

The glycosides are then recovered by elution using 100 ml of ethyl acetate. The composition of this fraction is studied by TLC, which enables the absence of free sugars to be verified (in this instance, at the end of the second fractionation on XAD-2).

The glycoside fraction was taken to dryness under vacuum and then taken up in 18 ml of water. The glycosidic extract used in the chromatographic and enzymatic experiments was thereby obtained.

HYDROLYSIS

The experimental protocols for the enzymatic hydrolysis on the glycosidic extract are collated in Table 3.

                                      TABLE 3                                      __________________________________________________________________________     Plan of the protocol for enzymatic hydrolysis of the glycosidic extract*            1st Stage          2nd Stage                                              __________________________________________________________________________          Addition of either                                                           50 μl of a Incubation 40° C., 16 h ADDITION of 50 μl of a                              solution of                                               solution of then β-glucosidase (0.15 nkat) to                             a-arabinosidase Extraction with pentane TESTS I and II                         (0.15 nkat) (5 × 250 μl) → GC                                  (TEST I)  Reincubation 40°, 16 h                                          then                                                                         or 50 μl of a 50 μl aqueous phase Extraction with pentane (5                                  × 250 μl) → GC                           200 μl of solution of silylation → GC                                glycosidic α-rhamnosidase  65 μl aqueous phase →                                     silylation → GC                                   extract** (0.15 nkat)                                                           (TEST II)  15 μl aqueous phase → TLC                                  12 μl aqueous                                                               phase → TLC                                                            OR                                                                             50 μl of a                                                                  solution of                                                                    β-glucosidase                                                             (0.15 nkat)                                                                    (TEST III)                                                                  __________________________________________________________________________      *The controls are carried out under the same conditions, in the presence       of the inactivated enzymes (95° C., 30 minutes).                        **Before the enzymatic hydrolyses, the glycosidic extract is washed again      several times with pentane in order to remove traces of terpenols.       

This hydrolysis is monitored by TLC and GC.

Monitoring of the Hydrolysis by TLC

The results are shown in FIG. 7: Monitoring by TLC of the enzymatic hydrolysis of the glycosidic extract.

1. Glycosidic extract

2. Glycosidic extract+β-glucosidase

3. Glycosidic extract+α-arabinosidase

4. Glycosidic extract+α-rhamnosidase

5. Glycosidic extract+α-arabinosidase+β-glucosidase

6. Glycosidic extract+α-rhamnosidase+β-glucosidase

The initial glycosidic extract shows a major spot (Rf 0.71, No. 2 on TLC) and two minor spots (Rf 0.67 and 0.79, No. 1 and No. 3 on TLC).

The hydrolysis of this extract with β-glucosidase gives rise to a faint spot having the same Rf (0.26) as glucose, and reduces the larger spot (No. 3).

In the case of the hydrolysis with α-arabinosidase, a substantial reduction is observed in the major spot (No. 2), and the appearance is noted of spots having Rf values (0.76, 0.79) identical to those of the terpene monoglucosides and with the Rf (0.31) of arabinose, and of a faint unknown spot with an Rf (0.55) less than that of rhamnose.

When the glycosidic extract is subjected to the action of 60 -rhamnosidase, the spot No. 1 corresponding to the Rf of Rha-Glc-Ger disappears and a faint spot with the Rf of the terpene monoglucosides appears. In this case, only rhamnose (Rf 0.59) is to be found as the monosaccharide liberated.

The consecutive action of β-glucosidase on each of the media incubated with either α-arabinosidase or α-rhamnosidase substantially reduces the spots with the Rf of monoglucosides, and gives rise to glucose.

However, since the major spot (No. 2) of the glycosidic extract did not completely disappear after sequential action of the glycosidases, the remaining portion was recovered and then subjected to an acid hydrolysis, under the conditions described below. Its hydrolysis at pH 3.0 neither gave rise to modification (verified by TLC). On the other hand, complete hydrolysis with 2 M trifluoroacetic acid caused this spot to disappear and liberated glucose, rhamnose and an unknown compound (Rf 0.55).

These TLC tests provided information about the fate of the carbohydrate portion of the glycosides during their hydrolysis with purified enzymes. It emerges from these results that the hydrolysis of terpene glycosides involves a sequential hydrolysis. The mechanism of this hydrolysis was demonstrated more precisely by GC analysis.

Monitoring of the Hydrolysis by GC

At each stage of the hydrolysis, the reaction products (sugars, terpene glycosides and terpenols) are analysed by GC. The results are shown in FIGS. 8 and 9:

FIG. 8: Monitoring by GC of the Enzymatic Hydrolysis of the Glycosidic Extract.

a. Silylated glycosidic extract

b. Glycosidic extract+α-arabinosidase

c. Glycosidic extract+α-arabinosidase+β-glucosidase

I.S. Internal Standard (Glc-pNP)

1 and 2. α- and β-arabinose

5 and 6. α- and β-glucose

a. Glc-Lin

b. Glc-Ner

c. Glc-Ger

A. Ara-Glc-Ner

B. Ara-Glc-Ger

FIG. 9: As FIG. 8, in the Case of the Sequential Hydrolysis by α-rhamnosidase+β-glucosidase.

3 and 4. α- and β-rhamnnose

The initial (silylated) glycosidic extract contains neither monosaccharides nor terpene monoglucosides (FIG. 8a).

The action of β-glucosidase does not substantially modify the profile of this extract, apart from the appearance of glucose. The latter, also observed by TLC during the monitoring of the hydrolysis of the glycosidic extract by this enzyme, hence probably does not originate from the hydrolysis of the terpene precursors, but from other glycosides.

The action of α-arabinosidase or of α-rhamnosidase gives rise to substantial modifications in respect of the profile of the glycosidic extract (FIGS. 8b and 9b). In particular, the two major peaks which have been identified (peak A=Ara-Glc-Ner; peak B=Glc-Ger) disappear completely after incubation of the medium with α-arabinosidase, while arabinose appears. The peak C, corresponding to the retention time of Rha-Glc-Ger, decreases after the action of α-rhamnosidase; the appearance of rhamnose is observed simultaneously.

Incubation of the glycosidic extract with each of these two glycosidases, gives rise, moreover, to three abundant monoglucosides (linalyl, neryl and geranyl glucosides), identified by comparison with reference substances. The larger part (77%) of these terpene monoglucosides is liberated by the action of α-arabinosidase, the remainder (23%) produced by the action of α-rhamnosidase (Table 4).

                  TABLE 4                                                          ______________________________________                                         Terpene monoglucosides liberated(1) by α-                                  arabinosidase and α-rhamnosidase from a glycosidic extract                                               % of mono-                                                                      α-arabinosidase α-rhamnosid                                       ase glucosides                               ______________________________________                                         Glc-Lin  10          9            19                                             Glc-Ner 28 5 33                                                                Glc-Ger 39 9 48                                                                % of mono-                                                                     glucosides 77 23  100                                                        ______________________________________                                          (1)The results are expressed as a percentage of the total amount of each       compound liberated by the two enzymes.                                   

The amounts of terpenols detected during this first stage of the hydrolysis, namely the hydrolysis of the glycosidic extract with each of the three glycosidases alone, were only negligible.

The consecutive action of β-glucosidase on the glycosidic extracts previously incubated either with α-arabinosidase or α-rhamnosidase (second stage) gives rise to the disappearance of the neryl and geranyl monoglucosides and a reduction in the linanyl monoglucoside, and the production of glucose (FIGS. 8c and 9c) and terpenols in large amounts.

It is known that the β-glucosidase used, which is extracted from sweet almond and selected for the purpose of studying the sequential hydrolysis of terpene glycosides, has a low affinity with respect to linalyl glucoside, resulting in incomplete hydrolysis of the latter under the conditions used.

The most abundant of the terpenols liberated are geraniol, nerol and linalool, in that order. Approximately 80% of these terpenols are derived from the conjugate action of α-arabinosidase an β-glucosidase.

Other terpenols or volatile compounds have also been detected, but in small amounts, after the sequential action of the enzymes, such as α-terpineol, citronellol, hydroxylinalools, linalool oxides (with the cis and trans furan-configurations and the cis pyran configuration), benzyl and phenyl ethyl alcohols, terpenepolyols, norisoprenoids (3-hydroxydamascone, 3-oxo-α-ionol), vinylgaiacol and ethylphenol.

The substantial modifications of the chromatographic profiles observed after the action of α-arabinosidase on the glycosidic extract led to an investigation in greater depth of the preponderant glycosides. For this purpose, fractions corresponding to the peaks A and B of the HPLC profile were collected. An aliquot portion was silylated and then analysed by GC. The correspondence between the peaks A and B of the two chromatograms of FIGS. 10 and 8a was thus established.

Legend to FIG. 10:

HPLC profile of the glycosidic extract

A. Ara-Glc-Ner

B. Ara-Glc-Ger

Furthermore, each fraction was subjected to the sequential action of α-arabinosidase and then β-glucosidase. At the end of each stage and according to the procedures described, the incubated medium was analysed by GC in order to test for sugars and glucosides on the one hand, and for terpenols on the other hand. The results are collated in Table 5. Thus, the peaks A and B were identified, respectively, as neryl and geranyl α-L-arabinofuranosyl-β-D-glucopyranosides. The identity of the geranyl glycoside was verified, in addition, by co-injection of the glycosidic extract with the synthetic compound.

                  TABLE 5                                                          ______________________________________                                         Identification of the peaks A and B collected by HPLC                                 Compounds appearing                                                       after action of                                                                              α-arabinosidase                                             and then Compounds                                                            α-arabinosidase β-glucosidase identified                          ______________________________________                                         Peak A arabinose +  glucose +    neryl                                            Glc-Ner nerol α-L-arabino                                                  furanosyl-β-D-                                                            glucopyrano-                                                                   side                                                                        Peak B arabinose + glucose + geranyl                                            Glc-Ger geraniol α-L-arabino                                               furanosyl-β-D-                                                            glucopyrano-                                                                   side                                                                      ______________________________________                                    

In another aspect, the action of the β-apiosidase on a glycosidic extract (FIG. 11A) was studied after the action of the Hemicellulase preparation (containing α-rhamnosidase, α-arabinosidase and β-glucosidase activities) on this extract. The Hemicellulase preparation eliminated the rhamnosyl- and arabinosylglucosides (FIG. 11B). Afterwards, the apiosylglucosides remaining were subjected to the hydrolytic action of the β-apiosidase and β-glucosidase of Klerzyme 200 (FIG. 11C). Noteworthy is the disappearance of the apiosylglucosides and the appearance of linalyl oxide glucosides, which demonstrate the sequential hydrolysis of the apiosylglucosides. The linalyl monoglucosides were not hydrolyzed, contrary to the geranyl and neryl monoglucosides. This is attributable to the weak affinity of the β-glucosidase of Klerzyme 200, vis-a-vis the linalyl glucosides.

These results collectively confirm that the enzymatic hydrolysis of grape terpene glycosides involves a sequential mechanism identical to that demonstrated on the synthetic substrates.

3--ENZYMATIC HYDROLYSIS ON NATURAL MEDIA

The enzymatic hydrolysis of a glycosidic extract incorporated, on the one hand into a natural medium (must or dry wine), and on the other hand into a reference medium (buffer), by the action of commercial preparations containing the requisite three glycosidases, was studied.

The hydrolysis protocol is as follows:

A juice and a wine of a vine variety not containing terpenols and terpene glycosides, but enriched beforehand with a known amount of glycosidic extract of muscat, are treated with commercial enzyme preparations. The media, as well as controls, are incubated at 25° C. for 86 hours (see the experimental protocol plan below). They are then passed through a column of Amberlite XAD-2 according to the protocol applied for the production of the glycosidic extract, and the terpenols are assayed by GC.

Plan of the Experimental Protocol

    ______________________________________                                                       + 0.5 ml Hemicellulase(1)                                          50 ml must + 0.5 ml Naringinase(2) Control                                     (pH 3.4: sugars 180 g/l) + 2 ml 2% NaN.sub.3 (3)                               or                                                                             Dry white wine + 0.5 ml Hemicellulase                                          (pH 3.1) + 0.5 ml Naringinase                                                   + 1 ml Glycosidic extract(4)                                                   + 2 ml 2% NaN.sub.3                                                            + 1 ml Glycosidic extract                                                       Control                                                                      50 ml buffer + 2 ml 2% NaN.sub.3                                               (50 mM citrate-                                                                phosphate) + 0.5 ml Hemicellulase                                              pH 3.3 + 0.5 ml Naringinase                                                      Reference                                                                     + 1 ml Glycosidic extract                                                      + 2 ml 2% NaN.sub.3                                                         ______________________________________                                          (1)0.5 ml of the solution (at a concentration of 15 mg/ml) of                  Hemicellulase REG2 used possessed 70 nkat of arabinosidase activity, 72        nkat of glucosidase activity and 9 nkat of rhamnosidase activity.              (2)0.5 ml of the solution (at a concentration of 5 mg/ml) of Naringinase       used possessed 78 nkat rhamnosidase activity and 0.1 nkat of glucosidase       activity.                                                                      (3)NaN.sub.3 is used in order to avoid microbial growth.                       (4)The glycosidic extract used is obtained by passing a must of Muscat         d'Alexandrie variety grapes through Amberlite XAD2. The amount of              glycosidic extract (1 ml) added in the tests corresponds to that found in      50 ml of must of Muscat d'Alexandrie variety grapes.                     

Under conditions resembling those found in oeno-logical practice, a liberation of terpenols is observed. This liberation is smaller in the case of must than in that of wine (Table 6). In terms of the percentage of aroma liberated after 86 hours at 25° C. compared with the amount liberated in a reference medium, this comes to 11% for the must and 28% for the wine. It may be noted that geraniol glycosides are more satisfactorily hydrolysed than those of linalool and nerol.

It was shown that the inhibition of the β-glucosidase activity by the glucose present in large amounts in the must can explain the lower efficiency relative to the wine.

The results are more satisfactory in the case of wine, since it is then possible, taking into account the preponderance of terpene glycosides, to at least double the free terpene fraction.

                  TABLE 6                                                          ______________________________________                                         Hydrolysis of a glycosidic extract incorporated                                  into a must and a wine by commercial enzyme preparations                            Compounds liberated relative to the reference medium(1) (%)                                               Benzyl Total                                   Linalool Nerol Geraniol alcohol terpenols                                    ______________________________________                                         MUST    9        6      15      46     11                                        WINE 14 18 49 50 28                                                          ______________________________________                                          (1)Reference: Glycosidic extract + citratephosphate buffer, pH 3.3 +           Hemicellulase REG2 + Naringinase.                                        

ACID HYDROLYSES

In order to detect the possible presence of terpene glycosides of unknown structure in the (TLC) spot resistant to enzymatic hydrolyses by the purified glycosidases, acid hydrolyses were applied to the compounds corresponding to this spot recovered by TLC.

1) The recovered medium (250 μl) is taken to dryness at 40° C. with a stream of nitrogen. 250 μl of 25 mM citratephosphate buffer (pH 3.0) are introduced into the flask, which is then sealed and left at 100° C. for 20 minutes. After being cooled, the medium is washed with pentane (5×250 μl). Internal standard is added to the pentane extract, which is then concentrated and analysed by GC. 25 μl of the aqueous phase are then placed on a TLC plate.

2) The recovered medium (250 μl), after being dried as above, is treated with 250 μl of 2 M trifluoroacetic acid and then left at 120° C. for 75 minutes. After being cooled, the medium is subjected to the same analyses as above.

4. Technological Application

The action of exogenous glycosidases of fungal origin has been studied during vinification of naturally sweet wines and of dry wines of the grape must (Muscat variety). Two enzymatic preparations, Hemicellulase and Klerzyme 200 (both produced by Gist-brocades) have been tested.

Experimental Protocol

Ripe, sound grapes of the Muscat of Frontignan variety were pressed. The must was sulfited (5 g/hl SO₂) and centrifuged. This was followed by the addition of the Hemicellulase enzyme preparation (containing (per liter must) 467 nkat α-arabinosidase, 296 nkat β-glucosidase and 31 nkat α-rhamnosidase) and 10 g/hl of a wine yeast (Saccharomyces cerevisiae, type K1, Institut Cooperatif du Vin, Maurin, France).

The naturally sweet wines are obtained after ending fermentation at mid-fermentation by the addition of alcohol until a final concentration of approximately of 17% (v/v) alcohol is reached.

For dry wines, vinification is continued until the exhaustion of the sugars.

The activity of the glycosidases were followed during the course of the fermentation. The glycosidases were isolated by precipitation with (NH₄)₂ SO₄ and afterwards were measured by use of the corresponding p-nitrophenyl glycosides.

After storing for 1.5 months at 20-22° C., the free and bound aroma component fractions of each sample were extracted on an Amberlite XAD-2 column in 50 ml aliquots, and analyzed via GC.

The exogenous glycosidases were effective in the liberation of aroma components in the vinification when there was no residual glucose in the medium (as in the case of dry wines). Thus, the content of terpenols (e.g. nerol, geraniol, citronellol, α-terpineol, hydroxy linalools) as well as norisoprenoid derivatives (β-damascenone, 3-hydroxy-β-damascone) increased in the dry wine originating from the enzymatically-treated must (Table 8). This analytical result was confirmed by taste panel. All of the tasters (15 in all) preferred the enzymatically-treated dry wine. The tasters showed no preference of the naturally sweet wines, whether enzymatically treated or not. The wines originating from the enzymatically-treated must were judged to be more aromatic (floral) and more typical.

In sweet wines originating from enzymatically-treated must, the hydrolysis of terpene diglycosides was not complete and was interrupted when only monoglucosides remained (FIG. 12). Noteworthy is the perceptible reduction of the major diglycosides (arabinosylglucosides, peaks 7 and 9) and a corresponding increase of the nerol and geraniol monoglucosides (peaks 2 and 3). The latter are not hydrolyzed in the naturally sweet wines due to the presence of glucose (30 g/l) which has the effect of inhibiting β-glucosidase (originating from Aspergillus niger) activity. On the other hand, these glucosides were hydrolyzed in the case of the enzymatically-treated dry wines.

However, a β-glucosidase (originating from the yeast Candida wickerhamii CBS 2928) has been able to hydrolyze neryl- and geranylmonoglucosides in the naturally sweet wines at a pH of 3.6 and above. This is due to the poor stability of the Candida wickerhamii β-glucosidase at a pH below 3.6.

At pH 3.6, both neryl- and geranylmonoglucosides present in the naturally sweet wines were hydrolyzed from 19% to 45% respectively. At pH 4.0, two thirds of each glucoside were hydrolyzed.

Contrary to arabinosyl- and rhamnosylglucosides, the apiosylglucosides are not hydrolyzed (FIG. 12, peaks 8 and 10) by the Hemicellulase enzymatic preparation. This is attributable to the absence of β-apiosidase activity in this preparation.

In another aspect, the action of an enzyme preparation (Klerzyme 200) containing β-apiosidase activity, in addition to α-rhamnosidase, α-arabinosidase and β-glucosidase activity, was studied during the fermentation of dry wines from the Muscat of Frontignan must. Klerzyme 200 (containing (per liter must) 2,770 nkat β-glucosidase, 704 nkat β-apiosidase, 276 nkat α-arabinosidase and 35 nkat α-rhamnosidase) and 10 g/hl of a wine yeast (Saccharoinvces cerevisiae, type K1) were added to the must.

The enzymatically treated dry wine was distinguished by the increase of the free aroma component fraction (Table 9), as compared with the case where the Hemicellulase preparation was used. Moreover, the presence of β-apiosidase activity in Klerzyme 200 permitted the hydrolysis of apiosylglucosides (Table 10). It is noteworthy that the linalyl glucoside and linalyl oxide glucosides, contrary to the other glucosides or glycosides, were not hydrolyzed. This may be explained by the weak activity of the β-glucosidase in the Klerzyme 200 preparation on these substrates.

Other volatile components such as vinyl, guaiacol and 3-oxo-α-ionol were detected in low concentrations in the enzymatically-treated wine.

The important increase of 2-phenylethanol in the wine is consequent from its synthesis by the yeast during the course of the fermentation of the must.

In another aspect, the exogenous glycosidases chosen have been found to be stable at the acidic pH of the must and of the wine.

                  TABLE 8                                                          ______________________________________                                         Components   Initial               Dry   Dry                                     (μg/l) Must NSW* NSW** wine* wine**                                       ______________________________________                                         Linalol, furanic                                                                            15      31      34    37    48                                      oxide, trans                                                                   Linalol, furanic 10 54 47 17 20                                                oxide, cis                                                                     Linalol, pyranic 347 313 326 366 382                                           oxide, trans                                                                   Linalol pyranic 90 79 88 100 98                                                oxide, cis                                                                     Linalol 1071 1157 1244 1314 1429                                               HO-trienol 9 169 221 163 142                                                   α-terpenol 40 225 256 333 472                                            Citronellol 29 43 63 65 218                                                    Nerol 40 32 61 50 321                                                          Geraniol 108 86 92 88 316                                                      E-8-hydroxy-linalol 44 Absent Absent Absent 230                                Z-8-hydroxy-linalol Absent Absent Absent Absent 300                            3,7-dimethyl-, 282 188 214 246 241                                             1,7-octanediene-3,6-diol                                                       3,7-dimethyl-, 1003 828 829 803 784                                            1,5-octanediene-                                                               3,7-diol                                                                       7-hydroxy- 17 51 54 59 55                                                      6,7-dihydrolinalol                                                             Total terpenols 3105 3256 3529 3641 5056                                       Others                                                                         β-damascenone Absent Absent Absent Absent 39                              3-hydroxy-β-damascone Absent Absent Absent Absent 121                     Benzyl alcohol 361 38 118 142 73                                               Z-phenylethanol 101 69203 68405 79574 77346                                  ______________________________________                                          NWS: Neutral Sweet Wine                                                        *Control                                                                       **Added with KLERZYME 200 (Gistbrocades)                                 

                  TABLE 9                                                          ______________________________________                                         Components     Initial                                                           (μg/l) Must NSW* NSW**                                                    ______________________________________                                         Linalol, furanic                                                                              Absent     55      136                                            oxide, trans                                                                   Linalol, furanic  91  61  91                                                   oxide, cis                                                                     Linalol, pyranic 333 356 389                                                   oxide, trans                                                                   Linalol pyranic  85  99  88                                                    oxide, cis                                                                     Linalol 1020  959 1089                                                         HO-trienol Absent  90  84                                                      α-terpenol  21 130 196                                                   Citronellol Absent  77 425                                                     Nerol  48  53 225                                                              Geraniol 173  61 300                                                           E-8-hydroxy-linalol  7  18 735                                                 Z-8-hydroxy-linalol Absent  37 497                                             3,7-dimethyl-, 190 237 360                                                     1,7-octanediene-3,6-diol                                                       3,7-dimethyl-, 1014  1127  1969                                                1,5-octanediene-                                                               3,7-diol                                                                       7-hydroxy- Absent  68 104                                                      6,7-dihydrolinalol                                                             Total terpenols 2982  3428  6671                                               Others                                                                         β-damascenone Absent Absent  62                                           3-hydroxy-β-damascone Absent Absent  57                                   Benzyl alcohol  95  48 174                                                     Z-phenylethanol 112 68131  55829                                             ______________________________________                                          NWS: Neutral Sweet Wine                                                        *Control                                                                       **Added with KLERZYME 200 (Gistbrocades)                                 

                  TABLE 1                                                          ______________________________________                                         Composition      Initial   Dry     Dry                                           μg/l Must wine* wine**                                                    ______________________________________                                         Linalyl glucoside and                                                                           323       292     429                                           linalyl furanic oxide                                                          Neryl glucoside 58 59 Absent                                                   Geranyl glucoside 67 70 Absent                                                 Linalyl rhamnosylglucoside 174  183  Absent                                    Neryl rhamnosylglucoside 92 89 Absent                                          Geranyl rhamnosylglucoside 73 73 Absent                                        Linalyl arabinosylglucoside 78 79 Absent                                       Neryl arabinosylglucoside 247  252  Absent                                     Geranyl arabinosylglucoside 263  231  Absent                                   Neryl apiosylglucoside 301  293  Absent                                        Geranyl apiosylglucoside 79 81 Absent                                          Benzyl apiosylglucoside 87 87 Absent                                           Geranic and neryl acidic 39 37 Absent                                          glycoside                                                                    ______________________________________                                          *Control                                                                       **Added with KLERZYME 200 (Gistbrocades)                                  

We claim:
 1. A process for obtaining a monoglycosidic aroma precursor from a diglycosidic aroma precursor, wherein said monoglycosidic aroma precursor comprises an aglycone-carbohydrate link bond and wherein said diglycosidic aroma precursor comprises, in addition, a β-apioside-carbohydrate link bond, comprising the step of enzymatically hydrolyzing the β-apioside-carbohydrate link bond with a composition that comprises at least a β-apiosidase, so as to liberate the monoglycosidic aroma precursor.
 2. A process for obtaining an aroma component or aroma from a diglycosidic aroma precursor that comprises a β-apioside-carbohydrate link bond and an aglycone-carbohydrate link bond, said method comprising the steps of: in a first stage according to claim 1, enzymatically hydrolysing the β-apioside-carbohydrate link bond with a composition that comprises at least a β-apiosidase so as to liberate a monoglycosidic aroma precursor; and in a second stage, enzymatically hydrolyzing the aglycone-carbohydrate link bond in said monoglycosidic aroma precursor obtained in said first stage with an enzyme capable of producing the aroma or aroma component therefrom, wherein said first and second stages involve use of one or more enzymes that are used simultaneously, or sequentially.
 3. A process according to claim 2 wherein said beta-apiosidase is a β-D-apiofuranosidase.
 4. A process according to claim 2 wherein said diglycosidic aroma precursor is present in a source selected from the group consisting of a fruit, an aromatic plant, a derivative of said fruit, a derivative of said aromatic plant, a byproduct of said fruit, a byproduct of said plant, and a plant material derived from an in vitro cell culture.
 5. A process according to claim 4 wherein the diglycosidic aroma precursor is found in must or wine.
 6. A process according to claim 4 wherein the diglycosidic aroma precursor is provide from an extract from which free terpenols and sugars have been removed, prior to said enzymatic hydrolysis step or steps.
 7. A process according to claim 1 wherein the diglycosidic aroma precursor is present in a natural medium.
 8. A process according to claim 1 wherein the diglycosidic aroma precursor is present in a synthetic substrate.
 9. A process according to claim 8 wherein the synthetic substrate is one to which a compound selected from the group consisting of:p-nitrophenyl α-L-rhamnopyranosyl-(1→>6)-β-D-glucopyranoside; geranyl α-L-rhamnopyranosyl-(1→22 6)-β-D-glucopyranoside; and p-nitrophenyl α-L-arabinofuranosyl-(1→>6)-β-D-glucopyranoside has been added.
 10. A process according to claim 2 wherein the enzymatic hydrolysis in the second stage thereof is performed by a β-glucosidase.
 11. A process according to claim 1 wherein said diglycosidic aroma precursor is selected from the group consisting of any precursor that comprises geraniol, linalool, nerol, α-terpineol, citronellol, a linalool oxide, a hydroxylinaool, phenyl ethyl alcohol, ethylphenol, benzylalcohol, 3-hydroxydamascone, 3-oxo-α-ionol, and vinylagaicol.
 12. A process for enhancing the aroma of a product, comprising the steps of obtaining an aroma according to the process of claim 2, and adding it to said product.
 13. A process for enhancing the aroma of a product, comprising the steps of:(a) adding to the product a diglycosidic aroma precursor, itself comprising a β-apioside-carbohydrate link bond and an aglycone-carbohydrate link bond; (b) enzymatically hydrolysing the β-apioside-carbohydrate link bond with a composition that comprises at least a β-apiosidase, so as to produce a monoglycosidic aroma precursor; and (c) enzymatically hydrolyzing the aglycone-carbohydrate link bond in the monoglycosidic aroma precursor so obtained with an enzyme capable of liberating the aroma or aroma component thereof.
 14. A process for enhancing the aroma of a product, comprising the steps of:(a) identifying a source material that contains a diglycosidic aroma precursor that comprises a β-apioside-carbohydrate link bond and an aglycone-carbohydrate link bond; (b) enzymatically hydrolysing the β-apioside-carbohydrate link bond of said diglycosidic aroma precursor with a composition that comprises at least a β-apiosidase so as to liberate a monoglycosidic aroma precursor; (c) adding said monoglycosidic aroma precursor to said product; and (d) enzymatically hydrolyzing the aglycone-carbohydrate link bond in said monoglycosidic aroma precursor after its addition to said product with an enzyme capable of producing the aroma or aroma component therefrom.
 15. A process for enhancing the aroma of a product, comprising the steps of:(a) identifying a source material that contains a diglycosidic aroma precursor that comprises a β-apioside-carbohydrate link bond and an aglycone-carbohydrate link bond; (b) enzymatically hydrolysing the β-apioside-carbohydrate link bond of said diglycosidic aroma precursor with a composition that comprises at least a β-apiosidase so as to liberate a monoglycosidic aroma precursor; (c) enzymatically hydrolyzing the aglycone-carbohydrate link bond in said monoglycosidic aroma precursor with an enzyme capable of producing the aroma or aroma component therefrom; and; (d) adding said aroma or aroma component to said product. 